1. Field of the Invention
Frost sensitive agricultural crops may be damaged by ice formation within their tissues. While water within the tissues may be supercooled to -5.degree. C. and below, the presence of certain natural epiphytic bacteria on the plant surface can promote the nucleation and formation of ice crystals at temperatures slightly below 0.degree. C. Such ice nucleation capable (INA.sup.+) bacteria are responsible for frost damage in a wide variety of important agricultural crops, such as corn, soybeans, wheat, tomatoes, deciduous fruit trees such as pear, almond, apple, cherry, and many subtropical plants such as citrus and avocado.
Many approaches have been developed for reducing such frost injury. Methods such as the use of wind machines to mix warm air from above with the cold air near the ground, the burning of fossil fuels to directly heat the air surrounding the crop plants, and the pumping of large amounts of water to release latent heat on or near crop plants are not highly effective, are costly, and can adversely affect the environment. Alternate methods for frost control are actively being sought.
One effective approach relies on the reduction of the natural epiphytic population of ice nucleation capable bacteria on the crop plants. Approaches include the use of bactericides, typically antibiotics, to reduce the total bacterial population; and the use of antagonistic, ice nucleation deficient (INA.sup.-) microorganisms which compete with members of the natural epiphytic flora. The antagonistic INA.sup.- strains heretofore have been selected from the natural populations of microorganisms which populate the plants, or have been generated by random mutagenesis of the naturally occurring INA.sup.+ strains. While use of such INA.sup.- mutants has enjoyed success, such randomly-induced mutations are not always stable and are often accompanied by other mutations which debilitate the strain and reduce its ability to compete with the INA.sup.+ strains.
It would thus be desirable to develop INA.sup.- strains from naturally-occurring INA.sup.+ bacteria, where the INA.sup.- strains are stable and free from other randomly-introduced mutations.
2. Description of the Prior Art
U.S. Pat. Nos. 4,045,910 and 4,161,084 describe the use of ice nucleation deficient microorganisms to inhibit frost injury. See also, copending application Ser. No. 294,604, filed Aug. 20, 1981. A number of papers have been published concerning the effect of bacteria on ice nucleation. See, for example, Lindow et al., Proc. Am. Phytopathol. Soc. (1977) 4.1976:169; Arny et al., Nature (1976) 262:282-283; Lindow et al. Phytophathology (1978) 68:523-528; Lindow et al., Appl. Environ. Microbiol. (1978) 36:831-838; Lindow et al., Proc. Am. Phytopathol Soc. (1977) 3.1976:224. Notice of an application for permission to construct and release Pseudomonas syringae pv. syringae and Erwinia herbicola carrying in vitro generated deletions of all or part of the genes involved in ice nucleation was published in the Federal Register [47 FR 41925] Sept. 22, 1982.